A complex quality control system (Endoplasmic Reticulum (ER) Associated Degradation;ERAD) degrades misfolded proteins as an integral part of the ER stress response and is central to a cell's ability to cope with stress. Growing evidence points to the importance of ER stress in tumor development and progression;yet, mechanisms underlying control of ER stress in normal and tumor cells are poorly understood. This application proposes to define a novel regulatory axis potentially controlling ER stress. In the current funded period we characterized the RING finger E3 ligase RNF5 and demonstrated that RNF5 is over-expressed in human cancer, specifically in breast cancer and melanoma. Inhibition of RNF5 in breast cancer cells reorganizes the cytoskeleton and sensitizes tumor cells to apoptosis following treatment with chemotherapeutic drugs, in a p53-dependent manner. Interestingly, RNF5 mediates both ubiquitination-mediated degradation and altered localization of substrates, including misfolded proteins. RNF5 is anchored via its C-terminal tail to the ER membrane where it interacts with a newly identified 7-transmembrane protein, JAMP. JAMP serves as a receptor for proteasome recruitment to the ER and is regulated by RNF5, indicating a novel regulatory axis in the ER stress response. While RNF5 limits JAMP activity, it promotes the degradation of misfolded proteins, by cooperating with Cl-HP or gp78, ubiquitin ligases implicated in ERAD. These findings position RNF5 as an important regulator of ERAD. Based on our preliminary results that (i) RNF5 is overexpressed in breast cancer and melanoma, (ii) RNF5 is required for breast cancer proliferation and cytoskeletal organization in a p53-dependent manner, and (iii) RNF5 limits JAMP activity and regulates the ER stress response, we have formulated the hypothesis that RNF5-JAMP regulation of ER stress contributes to breast cancer and melanoma proliferation and cytoskeletal organization which are required for their development. Our proposed studies will provide a mechanistic insight into the regulation of ER stress by the RNF5-JAMP module and its implications on breast cancer and melanoma development. To test our hypothesis, we will: (1) Determine the mechanism underlying upregulated RNF5 expression seen in breast cancer and melanoma. (2) Identify mechanism(s) underlying distinct RNF5 functions in ERAD (i) by cooperation with ER-associated ubiquitin ligases to ubiquitinate and degrade misfolded proteins and (ii) by control of JAMP recruitment of proteasomes to ER. (3) Assess the role of RNF5 in mammary tumor development using RNF5 knockout (KO) and inducible transgenic mice that we have generated and which will be crossed with mammary tumor model mice. Understanding RNF5-dependent regulation of ER stress via JAMP and its relationship to p53-dependent changes in breast cancer and melanoma should provide novel and important insight into mechanisms underlying control of ER stress in normal cells and in tumors.